The present invention relates to a microinjection apparatus, a trap plate and a microinjection method. More particularly, it relates to a microinjection apparatus, a trap plate and a microinjection method which can reliably inject a chemical agent into a cell and which can protect the distal end of a capillary needle.
In recent years, researches have been able to alter the genetic information of cells by injecting genes directly into the cells. Using this technique, the roles of the genes are clarified, and a tailor-made medical treatment, for example, a gene treatment suited to the genetic characteristics of an individual becomes possible.
As schemes for injecting the gene into the cell, there have been proposed an electrical method (eletroporation), a chemical method (lipofection), a biological method (vector method), a mechanical method (microinjection), an optical method (laser injection), etc. Disadvantageously, however, the electrical scheme damages the cell heavily because a cell membrane is broken by causing a large current to flow through the cell, the chemical scheme has an inferior efficiency because introducible genes are limited, and the biological method is incapable of ensuring safety because all materials cannot be introduced into the cell. On the other hand, the mechanical method appears to be the method which is the safest and which has the highest efficiency.
In the microinjection, as shown in FIG. 13, a capillary needle 10 is automatically moved to the position of a cell C so as to directly inject a chemical agent. On this occasion, the position of the cell C needs to be fixed within a laboratory dish 20 in order that the capillary needle 10 may move precisely to the position of the cell C. In FIG. 13, therefore, a trap plate 30 provided with a plurality of penetrating apertures is mounted in the laboratory dish 20, and the side of the trap plate 30 near to the bottom of the laboratory dish 20 is held at a negative pressure, whereby the cell C is sucked onto the penetrating aperture of the trap plate 30.
That is, in FIG. 13, the laboratory dish 20 is filled with a buffer liquid, and the trap plate 30 and the cell C are immersed in the buffer liquid. In addition, the buffer liquid is sucked from the bottom of the laboratory dish 20, whereby the side of the trap plate 30 near to the bottom of the laboratory dish 20 is brought to the negative pressure, and the cell C is sucked onto the penetrating aperture provided in the trap plate 30. Thus, the position of the cell C is fixed at the position of the penetrating aperture of the trap plate 30. When the coordinates of the penetrating aperture are stored beforehand, the capillary needle 10 can be moved precisely to the position of the cell C.
Meanwhile, in the case where the position of the cell C is fixed by the suction, it is difficult to appropriately adjust the relationship between the size of the cell and the size of the penetrating aperture. More specifically, as shown in FIG. 14 by way of example, in a case where the penetrating aperture of the trap plate 30 is excessively large relative to the size of the cell C, the cell C itself might be drawn into the penetrating aperture by the suction. Besides, as shown in FIG. 15 by way of example, in a case where the penetrating aperture of the trap plate 30 is excessively small relative to the size of the cell C, the position of the cell C cannot be reliably fixed, and the cell C sometimes undesirably moves when touched by the capillary needle 10.
In this regard, as described in Japanese laid-open Patent Publication No. 2005-318851, a penetrating aperture of small diameter is provided in a trap plate, while a recess of large diameter is provided around the penetrating aperture. That is, in the trap plate of Japanese laid-open patent publication No. 2005-318851, the penetrating aperture is provided at the center of the circular flat recess which is lower in level than the surroundings. According to such a trap plate, a cell is adsorbed on the trap plate by suction from the penetrating aperture, while at the same time, the position of the cell is reliably fixed by the recess.
In general, however, the cell membrane of a cell is highly flexible, and it is difficult to break even when a capillary needle is thrust thereagainst. This poses the problem that the reliable injection of a chemical agent into the cell is difficult. That is, as shown in FIG. 16 by way of example, even when the cell is reliably fixed by the circular recess and the penetrating aperture, the upside cell membrane sometimes stretches into the cell with the thrust of the capillary needle, so that the distal end of the capillary needle fails to enter the interior of the cell. In addition, even when the chemical agent is discharged from the distal end of the capillary needle in this state, it is not injected into the cell, and the microinjection ends in failure.
Besides, when the thrust magnitude of the capillary needle is increased to the extent that the upside cell membrane of the cell touches the downside cell membrane thereof as shown in FIG. 16, it is expected that the upside cell membrane will break, and that the distal end of the capillary needle will enter the interior of the cell. In this case, however, the capillary-needle distal end substantially abuts the downside cell membrane, and hence, a space sufficient for injecting the chemical agent thereinto is not defined between the capillary-needle distal end and the downside cell membrane.
Further, when the thrust magnitude of the capillary needle is excessively increased, the distal end of the capillary needle might pierce also the downside cell membrane, and the chemical agent might be discharged outside the cell. Moreover, the distal end of the capillary needle sometimes touches the trap plate and is then damaged. Especially in the microinjection, the cell to be handled has a size on the order of several μm to several tens μm, so that the movement of the capillary needle becomes minute. Therefore, even in case of an error of 1 to 2 μm by way of example, the capillary needle sometimes touches the trap plate, and it is likely to be damaged.
The present invention has been made in view of such drawbacks, and it has for its object to provide a microinjection apparatus, a trap plate and a microinjection method which can reliably inject a chemical agent into a cell and which can protect the distal end of a capillary needle.